The goal of this grant proposal is elucidation of the mechanisms regulating directed transport of polarized cytoplasmic components in developing Drosophila egg chambers. The polarized movement of RNAs, proteins, lipids and organelles underlies development, differentiation and cell function in many systems. Loss of polarity is associated with the onset of cell death or metastatic behavior in cells;thus, studying mechanisms cells employ to achieve polarity is important for understanding disease progression. We are exploiting Drosophila oogenesis as a model system to study polarized transport. Cytoplasm containing the maternal endowment of mRNA, proteins, and organelles is produced in nurse cells and continuously transported to growing oocytes through intercellular bridges or ring canals. Previous work on this project has revealed the importance of dynamically regulating the actin cytoskeleton in nurse cells to maintain the flow of cytoplasm to the oocyte. The focus has now shifted to examination of early transport, to understand how certain proteins are specifically enriched in the oocyte, in the context of a constant cytoplasm stream. This new phase of the project uses a collection of GFP-tagged endogenous proteins generated during the last grant period. By examining the distribution of these proteins during oogenesis, we have identified functional groups of proteins that are markedly enriched in the oocyte in the early stages of oocyte growth. Aim 1 of the proposal is to define and compare the transport of GFP-tagged proteins that are either oocyte enriched or ubiquitously distributed between nurse cells and oocytes. We will use live cell imaging and photobleaching techniques to determine the mobility of proteins associated with mRNA, ribosomes, endoplasmic reticulum or vesicles. We will also track subpopulations of proteins using fluorescence-based pulse-chase analysis. In Aim 2 we will test the involvement of the cytoskeleton by analyzing protein movement in mutant backgrounds that compromise actin or microtubules, and by incubating egg chambers in drugs that disrupt cytoskeletal organization. In Aim 3 we will map the sequences within proteins that are necessary and sufficient for targeting the proteins to oocytes, focusing initially on RNA-binding proteins important for escorting key mRNAs into the oocyte. We will test the functional consequences of mutant, non-targeted proteins on oocyte development. The polarized movement of RNAs, proteins, lipids and organelles underlies development, differentiation and cell function in innumerable systems including the nervous system and polarized epithelial cells. Loss of polarity is associated with the onset of cell death or metastatic behavior in cells;thus, studying mechanisms cells employ to achieve polarity is important for understanding disease progression. We are exploiting early development in the fruit fly as a model system to study polarized transport.